Glandular buccedaneum amd proc-



PM Oct. 19, 1937 UNITED STATES PATENT orrlce bosom GLANDULAB BUGUEDAN'EUM AND PROO- llllll,

Ohio; Louise K. Stewart, Kathleen Stew- L administrators of said Geo N. Stew d to said mag art, encased, arslgnors No Drawing. Appllcatio June 19 BerlalNo. 2823M} Adrenal gland tissue, as known, contains an agent variously designated as epinephrin, adrenalin, etc., having specifically a vaso-constrictor action. The function of the adrenal gland how- 5 ever is not limited to this, but is fundamentally more far-reaching; and where there is a destruction or removal of adrenal cortical tissue, systemic disturbances result, not correctable by administration of adrenalin, and some of which have for a long time been recogmzed as particular diseases or symptom complexes, although without identification of the ultimate cause. It is now found that conditions of this character are to be traced definitely to adrenal cortical insufficiencies. An agent readily suitable for use and effectively potent in adrenal insufiiciency is accordlngly fundamentally important and highly desirable.

To the accomplishment of the foregoing and related ends, the invention, then, consists of the features hereinafter fully described, and particularly pointed out in the claims, the following description setting forth by way of illustration a few of the various ways in which the principle of the invention may be applied.

If the cortical portion of adrenal glands be treated with an extractive solvent, there can be selectively derived an extractive or a material physiologically active in a manner referred to more in detail hereinafter. While the cortical tissue of the gland or interrenal tissue may be more or less completey isolated by being pared ofl as the initial source material, or in certain fishes this time may be had as an anatomically separate body or bodies called the interrenal gland, it is in practice usually more desirable to start with the whole glands of generally available abattoir source, for instance of sheep, beef or hogs, and extract and selectively separate out the 40 desired material (which for convenience we may call interrenalin). As solvents, glycerine, an

alcohol, such as ethyl alcohol, or 0.9% sodium chloride solution, or a solution of salts approximating the normal blood content and known familiarly as Ringers solution, may be employed.

Other solvents, such as acetone, chloroform or petroleum ether may also be used. The solvents may be used individually or in combination. Ordinarily, solvents containing a hydroxyl radical are more satisfactory, and these may include the various dilute aqueous salt solutions, glycerin and the alcohols, more specifically,

As an. example: Adrenal glands of sheep or beef, or pared cortical tissue therefrom, for instance, are thoroughly washed in distilled or pure 18 Claims. (Cl. 167-") water. and are then treated with about 5 to 10 parts of solvent to 1 by weight of the glandular material. The solvent may be added all at once, but it is preferable to start with a small amount, (about one cubic centimeter per gram of gland 6 tissue), and macerate and triturate the glandular material therewith. Additional volumes are then added with further trituration, until the entire quantity has been introduced. This is particularly desirable with combinations of alcohol and 10 glycerin. Starting for instance with a solvent consisting of 10% ethyl alcohol and 90% glycerin, in proportion to provide one cubic centimeter solvent per gram of gland material, an equal quantity of alcohol alone is added, with continued 15 trituration, and then successively further quantitles of alcohol, with continued trituration, until the alcohol content of the solvent mixture has been brought up to 90%. With the further additions of alcohol, some precipitation occurs. The 20 mixture is finally well shaken .in a mechanical shaker for 1 to 3 hours, and is then allowed to stand in a refrigeration. chamber to settle over a period of 6 to 12 hours. The liquid is then filbored of! through fibrous material, asbestos, filter 25 paper, or earthenware filters, with or without suction, the desired extractive material being present in the filtrate. The alcohol may now "be removed, by moderate heat if desired, for instance at 45 to 55 degrees C., and with or without a vac- 30 uum. Air or oxygen may be blown through the liquid, and this not only assists in disengaging the alcohol but has a further action in destroying contained adrenalin and probably other undesired constituents.

As another example: Sheep or beef adrenal glands or the cortical portions thereof, are washed thoroughly with distilled or pure water, and are then extracted with an aqueous medium, such as physiological salt solution made slightly alkaline, for example 0.1 to 1% of sodium bicarbonate may be added to the solvent. After thorough maceration and trituratlon, the mixture is centrifuged. Further extraction of the residue may then be carried on with glycerin and alcohol, the resulting extract added to the aqueous extract and the alcohol may be separated in whole or in part by blowing air or oxygen through the solution.

Products obtained as above are substantially free from adrenalin by the usualchemical and physiological tests.

' Instead of removing adrenalin by oxidation or by alkalies, it may be separated by less direct methods, so as to conserve it and thus make availin the raw material.

The possibility of inclusion of the adrenalin is minimized by employing the cortical portion of the adrenal gland when pared away from the medullary portion of the gland, this being most practicable in the case of the sheep adrenal.

The use of alkali and of alcohol in the procedure hereinbefore described has as one of its effects the removal of adrenalin and of objectionabie protein matter. It is readily apparent that other means for these results may be employed.

Potent extracts have also been prepared as follows: Pared cortical or interrenal tissue is triturated with glycerin in proportion to provide 4 to 5 cubic centimeters of glycerin per gram of tissue. The mixture is then thoroughly shaken up for several hours, for example, in a mechanical shaker. and allowed to stand in a refrigerator for 12 to 16 hours or longer. Alcohol is then added and the mixture filtered or it may be filtered first, the alcohol then added and again filtered. The volume of alcohol employed is 3 to 4 times that of the glycerin. The alcohol is then removed from the filtrate, for example, by gentle heating and aeration or in vacuo. The remaining glycerin extract may be administered undiluted via the alimentary tract or for intravenous administration may be diluted with physiological salt solution to produce an extractive of which 100 cubic centimeters may correspond. for example, to i to 2 grams of gland tissue.

The glycerated extractive obtained may be administered by the mouth, employing, if desired. capsules resistant to the gastric phase of digestion. although good results may be had without such capsules. For intravenous or subcutaneous administration, a glycerated extractive such as extractives corresponding to 1 to 2% of gland material extracted, is purified, sterilized, and diluted with sterile physiological normal salt solution, to contain 5 to 10% of the extractive. On iniectionin animals there is no discernible reaction, and in adrenal insufficiency there is a characteristic amelioration of the symptoms and decided prolongation of life. This is strikingly demonstrable in the case of adrenalectomizecl anmals.

In connection with each extract qualitative and some quantitative determinations of its potency may be secured. By a very large number of tests made upon completely adrcnaleetomized animals, we have determined their average and maximum periods of survival, which in the case of dogs are about one and two weeks respective- 1?. Potency determinations of the extractives may then advantageously be based on intravenous injection into a group of completely adrenalectomized animals, to standardized conditions, (say 12 to 20), the extractives increasing the average period, of survival of all the animals of the group, and increasing the period of survival of a substantial proportion, beyond the maximum of the untreated adrenalectomized animals, also ameliorating the symptoms of adrenal insuffieiency and reducing the severity of, and frequently entirely abolishing, the pathological condition of the alimentary canal found in untreated adrenalectomized animal's.

While a synthetic preparation of the agent may be a possibility, practically the preferable procedure is a selective derivation from available glandular substance. either by a special isolation of the tissue containing the desiredconable all of the respective glandular agents initially stltuent or by a general extraction and then a separation to obtain the extractive which inheres in the cortical portion of the gland in particular. In any instance, it will be seen that there is to be had a material which is characterized physiologicallyby its capability of relieving conditions due to adrenal cortical insufficiency, or an agent compensative of adrenal cortical deficiency generally.

Other modes of applying the principle of'the invention may be employed instead of those explained. change being made as regards the details disclosed. provided the features stated in any of the following claims or the equivalent of such be employed.

We therefore particularly point out and distinctly claim as our invention:

1. A process of making a physiologically potent agent remedial for adrenal cortical insufficiency and capable of prolonging the life of adrenalcctomized animals, which comprises triturating interrenal tissue with glycerin, adding alcohol, filtering the extractive-containing liquid from the residue, removing cpincphrin from, the liquid, and removing the alcohol.

2. A process of making an agent remedial for adrenal cortical insufficiency and capable of prolonging the life of adrenalectomized animals, which comprises triturating adrenal glands with a mixture of about 10% alcohol and glycerin, progressively adding further charges of alcohol, with trituration, to bring the alcoholic content up to about 90%, cooling and allowing to settle, filtering, removing the alcohol and oxygenatlng the solution.

8. A process of making an agent remedial for adrenal cortical insufficiency and capable of func-- tioning physiologically to prolong the life of adrenaleotomized animals, which comprises extracting moist adrenal glands with glycerine and a lesser amount of alcohol, removing epincphrin and constituents capable of giving a reaction on inJection of the extractives, and eliminating the alcohol.

4. As a remedial agent for adrenal cortical insufficiency, a physiologically potent material containing hormonal extractives from cortical tissue of adrenal glands and substantially free from adrenalin and adrenal gland extractives causing reaction on injection, and capable of functioning physiologically to prolong the life of adrenalectomized animals.

5. As a remedial agent for adrenal cortical insufficiency, a physiologically potent hormonal extract of fresh adrenal gland tissue substantially free from epinephrin and adrenal gland extractives causing reaction on injection and capable of functioning physiologically to prolong the life of adrenalectomized animals.

6. A glycerated solution containing hormonal extractives from cortical tissue of adrenal glands substantially free from epinephrin and adrenal gland extractives causing reaction on injection. said hormonal extractives being capable of functioning physiologically to prolong the life of adrenalectomined animals.

7. A process for producing an active hormonecontaining agent capable of functioning physiologically to prolong the life of adrenalectomized animals which comprises subjecting fresh adrenal tissue containing cortical portions thereof to the solvent action of glyeerine and alcohol, separating the resulting solution and subsequently removing epinephrin substantially completely therefrom.

Ill)

animals which comprises subjecting fresh adrenal tissue containing cortical portions thereof to the solvent action of a water soluble hydroxylated solvent of the class consisting of alcohol and glycerine and eliminating from said solution constituents not soluble in both said solvents and substantially all epinephrln.

9. An active hormonal extract of adrenal cortical tissue substantially completely soluble in alcohol and glycerine and substantially free from epinephrln and capable of functioning physiologically to prolong the life or adrenalectomized animals.

10. A solution derived from the adrenal glands comprising the active principal or hormone of the cortex of the adrenal glands and a solvent therefor which solution when hypodermically administered prolongs the life of adrenalectomized animals and is substantially free from epinephrln and adrenal gland extractives causing reaction on injection.

11. A solution derived from the adrenal glands comprising the active principal or hormone of the cortex of the adrenal glands and a dilute salt solution of salt concentration approximating that of the body fluids, which solution when hypodermically administered, ameliorates the symptoms of adrenal lnsufficiency and prolongs the life of adrenalectomized animals, said solution being free from epinephrln and adrenal gland extractives causing reaction on injection.

12. An extractive material derived from the adrenal glands comprising the active principal or hormone of the cortex of adrenal glands substantially free from epinephrin and adrenal gland extractives causing reaction on injection, which exthe cortical hormone, the steps which comprise tiiturating adrenal glands with glycerine and a lesser amount of alcohol, adding progressive charges of alcohol, filtering and oxygenating the filtrate, therebyforming a solution of said cortical hormone substantially free from epinephrln.

14. The process of making an extract contain ing the cortical hormone substantially free from epinephrln which comprises extracting adrenal glands with a solvent for the cortical hormone of the class consisting of dilute aqueous salt solution and glycerin, adding alcohol to the resulting solution, thereby separating alcohol insoluble constituents from said solution, and subsequently removing epinephrin substantially completely from the solution containing the cortical hor mone.

15. In process of making an extract containing the adrenal cortical hormone, the steps which comprise treating adrenal cortical tissue with an aqueous saline solution, adding alcoholic liquid to said solution, thereby removing protein from said solution, and subsequently substantially eliminating epinephrln from the solution whereby a solution is produced which is substantially free from epinephrln and from extractives which, on injection, produce a reaction.

16. In the process of obtaining and effecting purification of an extract containing the cortical hormone of suprarenal glands in. concentrated and potent form the steps which comprise separately employing upon material from adrenal cortical tissue containing the cortical hormone the solvent action of a dilute aqueous alkaline salt solution and of an alcoholic solution capable of preserving the activity thereof and having an alcohol concentration adapted to remove protein and other gland constituents capable of causing objectionable reactions, combining said solutions, and removing epinephrln from the resulting extract solution.

JULIUS M. ROGOFF. GEORGE N. STEWART. 

